Help with improving image

[IanW]

I have taken a break of a year and just returned - so am a bit rusty. Previously, I had been following the the threads on oblique illumination / VAC2 with interest, and here present a rather disappointing photograph using oblique illumination with polarisation. I think this might be a testate amoeba (perhaps others can decide). This image is actually composed of a stack (of 8 photos), but has some nasty artefacts.

This is a huge crop of the whole photograph - which in itself was taken with a X40 objective, projection lens X1.23, and optovar set to maximum.

Any guidance most welcome, thank you.




2020-11-26 16-40-58 (B,Radius8,Smoothing4) by Fortesmentum, on Flickr

[75RR]

I have taken a break of a year and just returned - so am a bit rusty.

Know what you mean, leaving off a given illumination technique for less time than that nevertheless has a toll.

I believe it was Beethoven who said "If I don't practice for one day - I can tell. If I don't practice for two days - my house-staff can tell. If I don't practice for three days my audience can tell.

[IanW]

That Beethoven quote has made my day. Thanks!

[DonSchaeffer]

what is wrong with this image? It looks great to me.

[micro]

Need more info on camera specs and more comparison pictures of default non oblique images and such

[MichaelG.]

what is wrong with this image? It looks great to me.

To put it bluntly, Don ... It has too many artefacts, and not enough real information.

MichaelG.

[mrsonchus]

Not a horrid image by any measure, but easily improved. As you are you have a somewhat chaotic mix of technique and consequentially results - no problem to improve that.

Personally, I'd say keep it simple and refine the basics first.

So, forget the polarisation, cropping, oblique, magnification etc and perfect or optimise the taking of an image with a correctly set-up Kohler-aligned system.
Perhaps begin with those base-line necessities to bring your technique to a starting point with the other add-ons, one at a time.

This approach has worked well for me with every single complex task I've encountered with my microscopy & photomicroscopy.

[MichaelG.]

This image is actually composed of a stack (of 8 photos), but has some nasty artefacts.

This is a huge crop of the whole photograph - which in itself was taken with a X40 objective, projection lens X1.23, and optovar set to maximum.

Any guidance most welcome, thank you.

.

To aid our ‘forensic analysis’ ... could you please post at least one example of the individual images that were used to make that stack.

Thanks
MichaelG.

[IanW]

Thank you for all observations with advice. MichaelG, I will post a couple of the single stacked images later, but for now, here are three single photos, which, in my opinion appear better than the stacked effort. I have yet to manage half reasonable stacked photos. These are uncropped images, neatened up in DXO.

Camera used is a Nikon d810 using mirror up and front curtain shutter.

DSC_0109_DxO by Fortesmentum, on Flickr

DSC_0107_DxO by Fortesmentum, on Flickr

DSC_0112_DxO by Fortesmentum, on Flickr

[MichaelG.]

Thanks for posting those, Ian

The blue one https://www.flickr.com/photos/67802150@N03/50661155997/ [predictably enough] looks the sharpest, but I’m sorry to say that even there, I can see no single detail that appears in sharp focus.

The stacking software will do the best it can ... but the principle is that it extracts the sharp details from each plane and combines them.

I think you need to discover why individual images are so ‘soft’ before worrying about the stacking.

Sorry if that appears harsh, but you did invite any guidance.

MichaelG.

[IanW]

Thanks Michael - appreciated. I agree, none look properly sharp, but not unexpected given that I am using the Optovar at full throttle - rather than moving up to a higher numerical aperture X60.

[apochronaut]

I was wondering about that. Isn't this just a case of empty magnification?

[MicroBob]

Camera used is a Nikon d810 using mirror up and front curtain shutter.

Hi Ian,
do you mean you used first elecronic shutter curtain function? Shutter shock can do bad things to image sharpness.

My experience with stacking: Contrary to my expectations stacking doesn't always increase image content. This only works when the number of images is high enough to have all relevant layers really sharp. 8 shots might not be enough, and placton life can move while taking the images. I would suggest to stick with individual images of interesting areas here.

Bob

[mrsonchus]

Also stacking can have too-many images if the subject is transparent/translucent and overlapping points of focus are included in the stack - I see this a lot with plant-section images also. As advised above, keep it simple and perfect technique first.

[micro]

How does the specimen look with your eyes when you use the eyepieces vs the camera?

[75RR]

I agree with mrsonchus, return to the basics and work your way back from there. Shouldn't take you long.

Good images as you know require getting all the ducks in a row, you probably just have a couple a little off - I would suggest you recheck Köhler, camera setup and use a Test Slide to avoid the many variables of a live subject.

[viktor j nilsson]

A micrometer slide (with cover glass) is also a boring, but very useful target when setting up your microscope. The flat, repeating pattern makes it very easy to tell if something is amiss.

[IanW]

Thanks to everyone who helped out.